Pyrrolidine and piperidine-2-carboxylic acid derivatives

ABSTRACT

New substituted acyl derivatives of amino acids which have the general formula ##STR1## are useful as angiotensin converting enzyme inhibitors.

SUMMARY OF THE INVENTION

This invention relates to new substituted acyl derivatives of aminoacids which have the general formula ##STR2## and salts thereof, whereinR₁ is hydroxy or lower alkoxy;

R₂ is hydrogen, lower alkyl or lower alkanoyl;

R₃ is hydrogen or lower alkyl;

R₄ is hydrogen, lower alkanoyl, benzoyl or ##STR3## A is hydrogen, loweralkyl or hydroxy-lower alkylene; B is hydrogen, lower alkyl, phenyl,phenyl-lower alkylene, hydroxy-lower alkylene, hydroxyphenyl-loweralkylene, amino-lower alkylene, guanidino-lower alkylene, mercapto-loweralkylene, lower alkyl-mercapto-lower alkylene, imidazolyl-loweralkylene, indolyl-alkylene, carbamoyl-lower alkylene or carboxy-loweralkylene; or A and B together form a (CH₂)_(p) bridge which completes aring of 5 or 6 atoms with the nitrogen and carbon to which they arejoined, one carbon optionally bearing a hydroxy group;

X is oxygen or sulfur;

n is 0 or 1;

m is 0, 1, 2, 3 or 4; and

p is 3 or 4. The asterisks denote centers of asymmetry.

DETAILED DESCRIPTION OF THE INVENTION

The invention in its broad aspects includes substituted acyl derivativesof amino acids having formula I above. The substituted acyl groups referto the side chains on the carbon beta to the nitrogen atom. The one sidechain has one or two sulfur containing groups and the second side chainhas one oxygen or sulfur containing group. Within the class defined byformula I, because of their properties, certain subgroups are preferred.

Compounds in the group represented by formula I which are derived fromor include the structure of the amino acids glycine, alanine, leucine,threonine, phenylalanine, lysine, arginine, glutamine, histidine,methionine, serine, cysteine, tyrosine, valine, asparagine, glutamicacid, proline, hydroxyproline, phenylglycine or tryptophane are broadlypreferred. Preferred modifications are compounds of formula I wherein R₁is hydroxy; R₂ is hydrogen, lower alkyl or lower alkanoyl (particularlyhydrogen, methyl or acetyl); R₃ is hydrogen or lower alkyl (particularlyhydrogen or methyl); R₄ is hydrogen, lower alkanoyl or benzoyl(particularly hydrogen, or acetyl); X is oxygen or sulfur; A ishydrogen; B is lower alkyl, guanidino-lower alkylene (particularlyguanidinopropyl), amino-lower alkylene (particularly amino-C₃ -C₄ -loweralkylene)or phenyl-lower alkylene(particularly phenylmethyl); or A and Bcomplete a 5- or 6-membered ring; m is 0 or 1 and n is 0 or 1. There isonly one alkyl substituent R₃.

Especially preferred are those compounds of formula I which are derivedfrom proline and have the formula ##STR4## The symbols have the samepreferred meanings described above.

The lower alkyl groups represented by any of the variables includestraight and branched chain hydrocarbon radicals from methyl to heptyl,for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,pentyl, isopentyl and the like. The lower alkylene groups are of thesame kind also having 1 to 7 carbons. Similarly, the lower alkoxy groupsare of the same kind with a link to oxygen, for example, methoxy,ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, t-butoxy and the like.The C₁ -C₄ members, especially C₁ and C₂ members, of all types arepreferred. Phenylmethyl is the preferred phenyl-lower alkylene group andmethoxy and t-butoxy the preferred lower alkoxy groups. The loweralkanoyl groups are the acyl radicals of the lower (up to 7 carbons)fatty acids, e.g., acetyl, propionyl, butyryl and the like, acetyl beingpreferred.

The products of formula I and the preferred subgroups can be produced byvarious methods of synthesis. According to a preferred method, the aminoacid of the formula ##STR5## wherein A, B and R₁ are defined as above,is acylated with an acid of the formula ##STR6## wherein R₂, R₃, R₄, X,m and n have the meaning defined above, by one of the known proceduresin which the acid IV is activated, prior to reaction with the amino acidIII, involving formation of a mixed anhydride, symmetrical anhydride,acid chloride, active ester, Woodward reagent K,N,N'-carbonylbisimidazole, EEDQ(N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) or the like. [For areview of these methods, see Methoden der Organischen Chemie(Houben-Weyl) Vol. XV, parts 1 and 2 (1974)].

When the product obtained is an ester, e.g., R₁ is t-butoxy, the estercan be converted to the free carboxy group by treatment withtrifluoroacetic acid and anisole, then with a base like sodiumbicarbonate. Conversely the free acid can be esterified by conventionalprocedures.

The acids of formula IV wherein n is 1 can be synthesized by severalprocedures. The preferred method is the addition of thiolacetic acid tothe substituted acrylic acid of the formula ##STR7##

When n is 0, the acids of formula IV are synthesized by the reaction ofan α-halo derivative of the formula ##STR8## with a thiolacetic acid orthiobenzoic acid to give the acid of the formula ##STR9## wherein R₆ islower alkyl, preferably methyl or phenyl.

An alternate procedure for the synthesis of compounds of formula Iwherein n is 0, is the acylation of the amino acid III with the acid offormula VI followed by displacement with thiolacetic acid or thiobenzoicacid.

The disulfides of formula I, wherein R₄ is ##STR10## are obtained byoxidation of the compound of the formula ##STR11## e.g., with analcoholic solution of iodine.

Another alternate method for the production of acids of formula IV isthe reaction of a halo derivative of the formula ##STR12## or theformula ##STR13## with thiolacetic acid or p-methoxybenzylmercaptan togive a compound of the formula ##STR14## or the formula ##STR15##respectively, wherein R₅ is lower alkanoyl, e.g., acetyl, ##STR16##After coupling of the acids XI or XII to the amino acid III, the loweralkanoyl group is removed by ammonolysis, e.g., with concentratedammonia, and the ##STR17## is removed by acidolysis, e.g., withtrifluoromethanesulfonic acid.

Also included in the invention are intramolecular disulfides wherein thetwo sulfur atoms, when X is sulfur, join in a dithiolane ring. Thesehave the formula ##STR18## wherein R₁, A and B have the same meaning asdefined previously. Such compounds are produced by acylating III with1,2-dithiolane-4-carboxylic acid as described above.

Products of formula I have two or three asymmetric carbon atoms. Thesecarbon atoms are indicated by an asterisk in formula I. The compoundsaccordingly exist in diastereoisomeric forms or in racemic mixturesthereof. All of these are within the scope of the invention. The abovedescribed syntheses can utilize the racemate or one of the enantiomersas starting material. When the racemic starting material is used in thesynthetic procedure, the stereoisomers obtained in the product can beseparated by conventional chromatographic or fractional crystallizationmethods. In general, the L-isomer with respect to the carbon of theamino acid constitutes the preferrred isomeric form.

The compounds of this invention form basic salts with various inorganicand organic bases which are also within the scope of the invention. Suchsalts include ammonium salts, alkali metal salts like sodium andpotassium salts (which are preferred), alkaline earth metal salts likethe calcium and magnesium salts, salts with organic bases, e.g.,dicyclohexylamine salt, benzathine, N-methyl-D-glucamine, hydrabaminesalts, salts with amino acids like arginine, lysine and the like. Thenon-toxic, phsiologically acceptable salts are preferred, although othersalts are also useful, e.g., in isolating or purifying the product.

The salts are formed in conventional manner by reacting the free acidform of the product with one or more equivalents of the appropriate baseproviding the desired cation in a solvent or medium in which the salt isinsoluble, or in water and removing the water by freeze drying. Byneutralizing the salt with an insoluble acid like a cation exchangeresin in the hydrogen form [e.g., polystyrene sulfonic acid resin -Dowex 50 (Mikes, Laboratory Handbook of Chromatographic Methods (VanNostrand, 1961) page 256] or with an aqueous acid and extraction with anorganic solvent, e.g., ethyl acetate, dichloromethane or the like, thefree acid form can be obtained, and, if desired, another salt formed.

Additional experimental details are found in the examples which arepreferred embodiments and also serve as models for the preparation ofother members of the group.

The compounds of this invention inhibit the conversion of thedecapeptide angiotensin I to angiotensin II and therefore are useful inreducing or relieving angiotensin related hypertension. The action ofthe enzyme renin on angiotensinogen, a pseudoglobulin in blood plasma,produces angiotensin I. Angiotensin I is converted by angiotensinconverting enzyme (ACE) to angiotensin II. The latter is an activepressor substance which has been implicated as the causative agent invarious forms of hypertension in various mammalian species, e.g., rats,dogs, etc. The compounds of this invention intervene in theangiotensinogen → angiotensin I →0 angiotensin II sequence by inhibitingangiotensin converting enzyme and reducing or eliminating the formationof the pressor substance angiotensin II.

The inhibition of the angiotensin converting enzyme by compounds offormula I can be measured in vitro with isolated angiotensin convertingenzyme from rabbit lungs following the procedure described by Cushmanand Cheung [Biochem. Parmacol., 20, 1637 (1971)], and with an excisedsmooth muscle assay [E O'Keefe, et al., Federation Proc. 31, 511 (1972)]in which these compounds have been shown to be powerful inhibitors ofthe contractile activity of angiotensin I and potentiators of thecontractile activity of bradykinin.

The administration of a composition containing one or a combination ofcompounds of formula I or physiologically acceptable salt thereof to thespecies of hypertensive mammal alleviates or reduces angiotensindependent hypertension. A single dose, or preferably two to four divideddaily doses, provided on a basis of about 5 to 1000 mg. per kilogram perday, preferably about 10 to 500 mg. per kilogram per day is appropriateto reduce blood pressure. The animal model experiments described by S.L. Engel, T. R. Schaeffer, M. H. Waugh and B. Rubin, Proc. Soc. Exp.Biol. Med. 143, 483 (1973) serve as a useful guide.

The substance is preferably administered orally, but parenteral routessuch as subcutaneously, intramuscularly, intravenously orintraperitoneally can also be employed.

The compounds of this invention can be utilized to achieve the reductionof blood pressure by formulating in compositions such as tablets,capsules or elixirs for oral administration or in sterile solutionsuspensions for parenteral administration. About 10 to 500 mg. of acompound or mixture of compounds of formula I or physiologicallyacceptable salt is compounded with a physiologically acceptable vehicle,carrier, excipient, binder, preservative, stabilizer, flavor, etc., in aunit dosage form as called for by accepted pharmaceutical practice. Theamount of active substance in these compositons or preparations is suchthat a suitable dosage in the range indicated in obtained.

The following examples are illustrative of the invention. Alltemperatures are in degrees celsius.

EXAMPLE 1 2-(Acetylthiomethyl)-3-(acetylthio)propanoic acid

A solution of 3.36 g. (40 mmoles) of thiolacetic acid in 40 ml. ofN-potassium hydroxide is added dropwise to a solution of2-bromomethyl-3-bromopropanoic acid in N potassium hydroxide (20 ml.).The mixture is stirred at room temperature overnight, acidified withconcentrated hydrochloric acid and extracted with ethyl acetate. Theorganic layer is dried and concentrated in vacuo. The residue isconverted into a dicyclohexylammonium salt (m.p. 116°-118°) and the saltconverted back into the free acid,2-(acetylthiomethyl)-3-(acetylthio)propanoic acid, by distributionbetween ethyl acetate and 10% potassium bisulfate.

EXAMPLE 2 1-[2-(Acetylthiomethyl)-3-(acetylthio)propanoyl]-L-prolinetert-butyl ester

2-(Acetylthiomethyl)-3-(acetylthio)propanoic acid (2.36 g.) is added toa solution of L-proline tert. butyl ester (1.71 g.),hydroxybenzotriazole (1.35 g.) and dicyclohexylcarbodiimide (2.06 g.) indichloromethane (15 ml.) chilled in an ice bath. The reaction mixture isstirred overnight at room temperature, the dicyclohexylurea is filteredoff, and the filtrate is washed neutral. The organic layer is dired, andconcentrated to dryness in vacuo to yield1-[2-acetylthiomethyl)-3-(acetylthio)propanoyl]-L-proline tert-butylester as a heavy oil (3.7 g.). Rf: 0.7 (silica gel-benzene, acetic acid7:1).

EXAMPLE 3 1-[(2-Acetylthiomethyl)-3-(acetylthio)propanoyl]-L-proline

(A) 1-[(2-Acetylthiomethyl)-3-(acetylthio)propanoyl]-L-prolinetert-butyl ester (b 2.7 g.) is dissolved in a mixture of trifluoroaceticacid and anisole and the mixture is stored at room temperature for onehour. The solvent is removed in vacuo and the residue is dissolved insaturated sodium bicarbonate solution. This aqueous solution isextracted with ethyl acetate, acidified with concentrated hydrochloricacid and reextracted with ethyl acetate. This second organic layer isdried and concentrated to dryness in vacuo. The residue ischromatographed on a column of silica gel with a mixture ofbenzene-acetic acid (7:1). The fractions containing the desired materialare pooled and concentrated to dryness to yield1-[(2-acetylthiomethyl)-3-(acethylthio)-propanoyl]-L-proline as an oil(1.3 g.). Rf: 0.3 (silica gel: benzene-acetic acid 75:25). (B) To asolution of L-proline (1.44 g.) and sodium carbonate (2.7 g.) in water(25 ml.) in an ice bath, 2-(acetylthiomethyl)-3-(acetylthio)propanicacid chloride (3.9 g. -- prepared from2-(acetylthiomethyl)-3-(acethythio)-propanoic acid and thionyl chloride)is added and the mixture is vigorously stirred at room temperature fortwo hours. After extraction with ethyl acetate, the aqueous layer isacidified and extracted with ethyl acetate. The organic layer is driedand concentrated to dryness. The residue,1-[(2-acetylthiomethyl)-3-(acetylthio)propanoyl]-L-proline ischromatographed as described in procedure (A) above.

EXAMPLE 4 1-(2-Mercaptomethyl-3-mercaptopropanoyl)-L-proline

1-[(2-Acetylthiomethyl-3-(acethylthio)propanoyl]-L-proline (1.2 g.) isdissolved in a mixture of water (12 ml.) and concentrated ammonia (12ml.) under an atmosphere of argon. After twenty minutes, the mixture isacidified with concentrated hydrochloric acid. The crystallineprecipitate 1-(2-mercaptomethyl-3-mercaptopropanoyl)-L-proline isfiltered and dried, yield 0.63 g., m.p. 138°-140°.

EXAMPLE 5 2,3-(Diacetylthio)propanoic acid

By substituting 2,3-dibromopropanoic acid for the2-bromomethyl-3-bromopropanoic acid in the procedure of Example 1,2,3-(diacetylthio)propanoic acid is obtained as an oil. Rf: 0.4 (silicagel:benzene-acetic acid 7:1).

EXAMPLE 6 1-[2,3-(Diacetylthio)propanoyl]-L-proline tert-butyl ester

By substituting 2,3-(diacetylthio)propanoic acid for the2-(acetylthiomethyl)-3-(acetylthio)propanoic acid in the procedure ofExample 2, 1-[2,3-(diacetylthio)-propanoyl]-L-proline tert-butyl esteris obtained as an oil. Rf: 0.5 (silica gel:chloroform-methane 98:2).

EXAMPLE 7 1-[2,3-(Diacetylthio)propanoyl]-L-proline

By substituting 1-[2,3-(diacetylthio)propanoyl]-L-proline tert-butylester for the 1-[(2-acetylthiomethyl)-3-acetylthiopropanoyl]-L-prolinetert-butyl ester in the procedure of Example 3A,1-[2,3-(diacetylthio)propanoyl]-L-proline is obtained. Rf: 0.45 (silicagel: benzene: acetic acid, 75-25).

EXAMPLE 8 1-[2,3-Dimercaptopropanoyl]-L-proline

By substituting 1-[2,3-(diacetylthio)propanoyl]-L-proline for the1[(2-acetylthiomethyl-3-(acetylthio)-propanoyl]-L-proline in theprocedure of Example 4, 1-[2,3-dimercaptopropanoyl]-L-proline isobtained. This product is isolated as an oil by extraction with ethylacetate after acidification of the reaction mixture. Rf: 0.43 (silicagel-no indicator-benzene:acetic acid 75:25).

EXAMPLE 9 2-Bromo-3-(4-methoxybenzylmercapto)propanoic acid

By substituting 3-(4-methoxybenzyl)cysteine for the S-benzylcysteine inthe procedure described by N. Izumiya (Chemical Abstracts, 47, 3236),2-bromo-3-(4-methoxybenzylmercapto)propanoic acid is obtained.

EXAMPLE 10 3-(4-Methoxybenzylmercapto)-2-(methylthio)propanoic acid

A solution of 2-bromo-3-(4-methoxybenzylmercapto) propanoic acid (15g.), methylmercaptan (3 g.) and sodium hydroxide (4.6 g.) in 95% ethanol(25 ml.) is refluxed for twelve hours. The sodium bromide is filteredoff and the filtrate is concentrated to dryness in vacuo. The residue isdissolved in water, acidified with concentrated hydrochloric acid andextracted with ethyl acetate. The organic layer is dried andconcentrated to dryness to yield3-(4-methoxybenzylmercapto)-2-(methylthio)propanoic acid.

EXAMPLE 111[3-(4-Methoxybenzylmercapto)-2-(methylthio)propanoyl]-L-prolinetert-butyl ester

By substituting 3-(4-methoxybenzylmercapto)-2-(methylthio)propanoic acidfor the 2-(acetylthiomethyl)-3-(acetylthio)propanoic acid in theprocedure of Example 2,1-[3-(4-methoxybenzylmercapto)-2-(methylthio)propanoyl]-L-prolinetert-butyl ester is obtained.

EXAMPLE 12 1-[3-Mercapto-(2-methylthio)propanoyl]-L-proline

1-[3-(4-Methoxybenzylmercapto)-2-(methylthio)propanoyl]-L-prolinetert-butyl ester (0.5 g.) is dissolved in a mixture of dichloromethane(2 ml.) and anisole (1.1 ml.) under a blanket of argon in an ice bath.Trifluoromethanesulfonic acid (1 g.) is added and the mixture is stirredat room temperature for thirty minutes. The solvent is removed in vacuoand the residue is distributed between water and ethyl acetate. Theorganic layer is washed six times with water, dried and concentrated todryness in vacuo to yield1-[3-mercapto-2-(methylthio)propanoyl]-L-proline.

EXAMPLE 13 Ethyl-2-methylthiomethylacrylate

A solution of methylmercaptan 4.8 g.) and sodium (2.3 g.) in absoluteethanol (75 ml.) is added dropwise to a solution ofethyl-2-bromomethylacrylate (19.3 g.) in ethanol (25 ml.). After twohours, it is diluted with water (400 ml.) and extracted with methylenechloride. The organic layer is dried and concentrated to dryness invacuo to yield ethyl-2-methylthiomethylacrylate.

EXAMPLE 14 (2-Methylthiomethyl)acrylic acid

Ethyl-2-methylthioacrylate (5 g.) is heated with 10% sulfuric acid (100ml.) at 80° for one hour. After cooling, the aqueous phase is extractedwith ethyl acetate to give (2-methylthiomethyl)acrylic acid.

EXAMPLE 15 3-(Acetylthio)-2-(methylthiomethyl)propanoic acid

A mixture of 2-(methylthiomethyl)acrylic acid (5.5 g.) and thiolaceticacid (5 ml.) is heated in the steam bath until disappearance of vinylproton absorption in the nmr. The mixture is concentrated to remove theexcess thiolacetic acid to obtain3-(acetylthio)-2-(methylthiomethyl)propanoic acid.

EXAMPLE 16 1-(3-Acetylthio)-2-methylthiomethyl)propanoyl]-L-proline

To a solution of L-proline (1.44 g.) and sodium carbonate (2.7 g.) inwater (25 ml.) in an ice bath,3-(acetylthio)-2-(methylthiomethyl)propanoic acid chloride (preparedfrom the acid of Example 15 with thionyl chloride) (3.6 g.) is added,and the mixture is vigorously stirred at room temperature for two hours.After extraction with ethyl acetate, the aqueous layer is acidified andextracted with ethyl acetate. The organic layer is dried andconcentrated to dryness in vacuo to give1-[3-(acetylthio)-2-methylthiomethyl)propanoyl]-L-proline.

EXAMPLE 17 1-[3-Mercapto-2-(methylthiomethyl)propanoyl]-L-proline

1-[3-(acetylthio)-2-(methylthiomethyl)propanoyl]-L-proline (1.2 g.) isdissolved in a mixture of water (12 ml.) and concentrated ammonia (12ml.) under a blanket of argon. After twenty minutes, the reactionmixture is acidified and extracted with ethyl acetate. The organic layeris dried and concentrated to dryness to yield1-([3-mercapto-2-(methylthiomethyl)propanoyl]-L-proline.

EXAMPLE 18 2-Acetoxymethyl-3-(acetylthio)propanoic acid

(A) By substituting 2-(acetoxymethyl)acrylic acid [J. Org. Chem., 28,2835 (1963)] for the 2-(methylthiomethyl)acrylic acid in the procedureof Example 15, 2-acetoxymethyl-3-(acetylthio)propanoic acid is obtained.

(B) By substituting 2-(hydroxymethyl)acrylic acid [J. Org. Chem., 28,2835 (1963)] for the 2-(methylthiomethyl)acrylic acid in the procedureof Example 15, and then submitting the product to acetylation withacetic anhydride, 2-hydroxymethyl-3-(acetylthio)propanoic acid and2-acetoxymethyl-3-(acetylthio)propanoic acid are obtained.

EXAMPLE 19 1-[2-(Acetoxymethyl)-3-(acetylthio)propanoyl]-L-proline

By substituting 2-(acetoxymethyl)-3-(acetylthio)-propanoic acid for the3-acetylthio-2-methylthiomethylpropanoic acid in the procedure ofExample 6, 1-[2-(acetoxymethyl)-3-(acetylthio)propanoyl]-L-proline isobtained.

EXAMPLE 20 1-(2-Hydroxy-3-mercaptopropanoyl)-L-proline

1-[2-acetoxymethyl-3-(acetylthio)propanoyl]-L-proline (1.5 g.) isdissolved in a mixture of water (12 ml.) and concentrated ammonia (12ml.) under a blanket of argon. After one hour, the reaction mixture isconcentrated to ca. dryness, diluted with water and the solution appliedto a column of cation exchange resin (Dowex 50) in the hydrogen cycle.The water eluate is concentrated to small volume and freeze dried toyield 1-(2-hydroxymethyl-3-mercaptopropanoyl)-L-proline.

EXAMPLE 21 2-Acetylthiomethyl-5-oxo-6-methylheptanoic acid

By substituting 2-methylene-5-oxo-6-methylheptanoic acid [Agr. Biol.Chem., 37, 2365, (1973)] for the (2-methylthiomethyl)acrylic acid in theprocedure of Example 15, 2-acetylthiomethyl-5-oxo-6-methylheptanoic acidis obtained.

EXAMPLE 22 1-[2-(Acetylthiomethyl)-5-oxo-6-methylheptanoyl]-L-prolinetert.butyl ester

By substituting 2-acetylthiomethyl-5-oxo-6-methylheptanoic acid for the2-(acetylthiomethyl)-3-acetylthiopropanoic acid in the procedure ofExample 2, 1-[2-acetylthiomethyl)-5-oxo-6-methylheptanoyl]-L-prolinetert.butyl ester is obtained.

EXAMPLE 23 1-[2-(Acetylthiomethyl)-5-oxo-6-methylheptanoyl]-L-proline

By substituting1-[2-(acetylthiomethyl)-5-oxo-6-methylheptanoyl]-L-proline tert.butylester for 1-[2-(acetylthiomethyl)-3-acetylthiopropanoyl]-L-prolinetert.butyl ester in the procedure of Example 3,1-[2-(acetylthiomethyl)-5-oxo-6-methylheptanoyl]-L-proline is obtained.

EXAMPLE 24 1[2-(Acetylthiomethyl)-5-hydroxy-6-methylheptanoyl]-L-proline

1-[2-acetylthiomethyl)-5-oxo-6-methylheptanoyl]-L-proline (1.5 g.) isdissolved in cold methanol (10 ml.) and sodium borohydride (0.12 g.) isadded. After two hours the reaction mixture is diluted with water (100ml.) acidified with concentrated hydrochloric acid and extracted withethyl acetate. The organic layer is dried and concentrated to dryness invacuo to give1-[2-(acetylthiomethyl)-5-hydroxy-6-methylheptanoyl]-L-proline.

EXAMPLE 25 1-[2-Mercaptomethyl-5-hydroxy-6-methylheptanoyl]-L-proline

By substituting1-[2-(acetylthiomethyl)-5-hydroxy-6-methylheptanoyl]-L-proline for the1-3-acetylthio-2-(methylthiomethyl)propanoyl]-L-proline in the procedureof Example 17,1-[2-mercaptomethyl-5-hydroxy-6-methylheptanoyl]-L-proline is obtained.

EXAMPLE 26 1-[2-Benzoylthio-3-methoxybutanoyl]-L-proline

To a solution of L-proline (5.75 g.) in N sodium hydroxide (50 ml.)chilled in an ice bath, 2N sodium hydroxide (25 ml.) and2-bromo-3-methoxybutyric acid chloride [obtaind from2-bromo-3-methoxybutyric acid [J. Am. Chem. Soc., 71, 1096, (1949)] andthionyl chloride] (10.7 g.) are added, with vigorous stirring. Afterthree hours, thiobenzoic acid (7.5 g.) and potassium carbonate (4.8 g.)are added and the mixture is stirred at room temperature overnight. Thereaction mixture is acidified and extracted with ethyl acetate. Theorganic layer is concentrated to dryness and the residue ischromatographed on a column of silica gel with benzene-acetic acid toyield 1-[2-benzoylthio-3-methoxybutanoyl]-L-proline.

EXAMPLE 27 1-[2-Mercapto-3-methoxybutanoyl]-L-proline

By substituting 1-[2-benzoylthio-3-methoxybutanoyl]L-proline for the1-[3-acetylthio-2-(methylthiomethyl)-propanoyl]-L-proline in theprocedure of Example 17, 1-[2-mercapto-3-methoxybutanoyl]-L-proline isobtained.

EXAMPLE 28 1-[2-Benzoylthio-3-(methylthio)propanoyl]-L-proline

By substituting 2-bromo-3-(methylthio)propanoic acid (ChemicalAbstracts, 47, 3236) for the 2-bromo-3-methoxybutyric acid in theprocedure of Example 26,1-[2-benzoylthio-3-(methylthio)propanoyl]-L-proline is obtained.

EXAMPLE 29 1-[2-Mercapto-3-(methylthio)propanoyl]-L-proline

By substituting 1-[2-benzoylthio-3-(methylthio)-propanoyl]-L-proline forthe 1-[3-acetylthio-2-(methylthiomethyl)propanoyl]-L-proline in theprocedure of Example 17,1-[2-mercapto-3-(methylthio)propanoyl]-L-proline is obtained.

EXAMPLE 301,1-[Dithiobis[2-(methylthiomethyl)-3-propanoyl]]-bis-L-proline

1-[3-mercapto-2-(methylthiomethyl)propanoyl]-L-proline (2.0g.) isdissolved in 40 ml. of 0.5 N sodium hydroxide, and an ethanolic solutionof iodine is added until persistent yellow color, while keeping the pHbetween 5 and 7 by careful addition of N sodium hydroxide. The yellowcolor is discharged with a small amount of sodium thiosulfate, and afteracidification with concentrated hydrochloric acid, the aqueous layer isextracted with ethyl acetate. The organic layer is dried andconcentrated to dryness to yield1,1'-[dithiobis[2-(methylthiomethyl)-3-propanoyl]]-bis-L-proline.

EXAMPLE 31 1,1'-[Dithiobis(2-hydroxymethyl-3-propanoyl)]-bis-L-proline

By substituting 1-(2-hydroxymethyl-3-mercaptopropanoyl)-L-proline forthe 1-[3-mercapto-2-(methylthiomethyl)propanoyl]-L-proline in theprocedure of Example 30,1,1'-[dithiobis-(2-hydroxymethyl-3-propanoyl)]-bis-L-proline isobtained.

EXAMPLE 32 1-[2-Mercaptomethyl-3-mercaptopropanoyl]-pipecolic acid

By substituting pipecolic acid tert.butyl ester for the L-prolinetert.butyl ester in the procedure of Example 2, and then submitting theproduct to the procedures of Examples 3 and 4,1-[2-(acetylthiomethyl)-3-(acetylthio)propanoyl]-pipecolic acidtert.butyl ester,1-[(2-acetylthiomethyl)-3-(acetylthio)propanoyl]pipecolic acid and1-[2-mercaptomethyl-3-mercaptopropanoyl]pipecolic acid are obtained.

EXAMPLE 33 1-[2-Hydroxymethyl-3-mercaptopropanoyl]pipecolic acid

By substituting pipecolic acid for the L-proline in the procedure ofExample 19, and then submitting the product to the procedure of Example20, 1-[2-acetoxymethyl-3-(acetylthio)propanoyl]pipecolic acid and1-[2-hydroxymethyl-3-mercaptopropanoyl]pipecolic acid are obtained.

EXAMPLE 34 N-[2-(Mercaptomethyl)-3-mercaptopropanoyl]-L-leucine

By substituting L-leucine for the L-proline in the procedure of Example3B, and then submitting the product to the procedure of Example 4,1-[(2-acetylthiomethyl)-3-acetylthiopropanoyl]-L-leucine and1-[(2-mercaptomethyl)-3-mercaptopropanoyl]-L-leucine are obtained.

EXAMPLE 35 N-[2-(Mercaptomethyl)-3-mercaptopropanoyl]-L-phenylalanine

By substituting L-phenylalanine for the L-proline in the procedure ofExample 3B, and then submitting the product to the procedure of Example4, 1-[2-acetylthiomethyl)-3-acetylthiopropanoyl]-L-phenylalanine and1-[2-(mercaptomethyl)-3-mercaptopropanoyl]-L-phenylalanine are obtained.

EXAMPLE 36 N.sup.α-[2-(Acetylthiomethyl)-3-(acetylthio)propanoyl]-L-arginine

L-Arginine (8.7 g.) is dissolved in aqueous N sodium hydroxide (50 ml.)and the solution is chilled in an ice bath with stirring. 2N sodiumhydroxide (25 ml.) and 2-(acetylthiomethyl)-3-acetylthiopropanoic acidchloride (15.6 g.) are added in that order, and the mixture is removedfrom the ice bath and stirred at room temperature for two hours. Thereaction mixture is extracted with ethyl acetate and the aqueous layeris applied to a cation exchange column (Dowex 50) in the hydrogen cycle.After washing with water to elute acidic material, the N.sup.α-[2-(acetylthiomethyl)-3-(acetylthio)propanoyl]-L-arginine is elutedwith a pyridineacetic acid buffer at pH 6.5.

EXAMPLE 37 N.sup.α -[2-Mercaptomethyl)-3-mercaptopropanoyl]-L-arginine

N.sup.α -[2-(acetylthiomethyl)-3-(acetylthio)propanoyl]-L-arginine (1g.) is dissolved in a mixture of water (5 ml.) and concentrated ammonia(5 ml.). After thirty minutes, the solution is concentrated to drynessand the residue is applied to a column of Dowex 50 in the hydrogencycle. After washing with water to remove acidic material, the N.sup.α-[2-mercaptomethyl-3-mercaptopropanoyl]-L-arginine is eluted with apyridine-acetic acid buffer at pH 6.5.

EXAMPLE 38 N.sup.α-[2-(Acetylthiomethyl)-3-(acetylthio)propanoyl]-N.sup.ε -tert.butyloxycarbonyl-L-lysine tert.butyl ester

By substituting N.sup.ε -tert.butyloxycarbonyl-L-lysine tert.butyl esterfor the L-proline tert.butyl ester in the procedure of Example 2,N.sup.α -[2-(acetylthiomethyl)-3-(acetylthio)propanoyl]-N.sup.ε-tert.butyloxycarbonyl-L-lysine tert.butyl ester is obtained.

EXAMPLE 39 N.sup.α-[2-(Acetylthiomethyl)-3-(acetylthio)propanoyl]-L-lysine

N.sup.α -[2-(acetylthiomethyl)-3-(acetylthio)propanoyl]-N.sup.ε-tert.butyloxycarbonyl-L-lysine tert.butyl ester (3 g.) is dissolved ina mixture of trifluoroacetic acid and anisole, and the mixture is storedat room temperature for one hour. The trifluoroacetic acid is removed invacuo, the residue is distributed between ether and water and theaqueous layer is applied to a column of Dowex 50. After washing theacidic material with water, the N.sup.α-[2-(acetylthiomethyl)-3-(acetylthio)propanoyl]-L-lysine is eluted witha buffer of pyridineacetic acid at pH 6.5.

EXAMPLE 40 N.sup.α -[2-Mercaptomethyl-3-mercaptopropanoyl]-L-lysine

By substitutingN-[2-(acetylthiomethyl)-3-(acetylthio)-propanoyl]-L-lysine for N.sup.α-[2-(acetylthiomethyl)-3-(acetylthio)-propanoyl]-L-arginine in theprocedure of Example 37, N.sup.α-[2-mercaptomethyl-3-mercaptopropanoyl]-L-lysine is obtained.

EXAMPLE 41 N-[2-(Mercaptomethyl)-3-mercaptopropanoyl]glycine

By substituting glycine for the L-proline in the procedure of Example 3Band then submitting the product to the procedure of Example 4,N-[2-(acetylthiomethyl)-3-(acetylthio)propanoyl]glycine andN-[2-(mercaptomethyl)-3-mercaptopropanoyl]glycine are obtained.

EXAMPLE 42 N-[2-(Mercaptomethyl)-3-mercaptopropanoyl]-L-glutamine

By substituting L-glutamine for the L-proline in the procedure ofExample 3B, and then submitting the product to the procedure of Example4, N-[2-(acetylthiomethyl)-3-(acetylthio]propanoyl-L-glutamine and1-[2-(mercaptomethyl)-3-mercaptopropanoyl]-L-glutamine are obtained.

EXAMPLE 43 N-[2-(Mercaptomethyl)-3-mercaptopropanoyl]-L-threonine

By substituting L-threonine for the L-proline in the procedure ofExample 3B, and then submitting the product to the procedure of Example4, N-[2-(acetylthiomethyl)-3-(acetylthio)propanoyl]-L-threonine and1-[2-(mercaptomethyl)-3-mercaptopropanoyl]-L-threonine are obtained.

EXAMPLE 441-[2-(Mercaptomethyl)-3-mercaptopropanoyl]-4-hydroxy-L-proline

By substituting 4-hydroxy-L-proline for the L-proline in the procedureof Example 3B, and then submitting the product of the procedure ofExample 4,1-[2-(acetylthiomethyl)-3-acetylthio)propanoyl]-4-hydroxy-L-proline and1-[2-(mercaptomethyl)-3-mercaptopropanoyl]-4-hydroxy-L-proline areobtained.

EXAMPLE 45 N.sup.α -[2-(Mercaptomethyl)-3-mercaptopropanoyl]-L-histidine

By substituting L-histidine for the L-arginine in the procedure ofExample 36 and then submitting the product to the procedure of Example37, N.sup.α -[2-(mercaptomethyl)-3-mercaptopropanoyl]-L-histidine isobtained.

EXAMPLE 46 N-[2-(mercaptomethyl)-3-mercaptopropanoyl]-L-tryptophane

By substituting L-tryptophane for the L-proline in the procedure ofExample 3B and then submitting the product to the procedure of Example4, N-[2-(acetylthiomethyl)-3-(acetylthio)propanoyl]-L-tryptophane andN-[2-(mercaptomethyl)-3-mercaptopropanoyl]-L-tryptophane are obtained.

EXAMPLE 47 N.sup.α -[2-(Hydroxymethyl)-3-mercaptopropanoyl]-L-arginine

By substituting 2-(acetoxymethyl)-3-(acetylthio)-propanoic acid chloridefor the 2-(acetylthiomethyl)-3-(acetylthio)propanoic acid chloride inthe procedure of Example 36, and then submitting the product to theprocedure of Example 37, N.sup.α-[2-(acetoxymethyl)-3-(acetylthio)-propanoyl]-L-arginine, and N.sup.α-[2-(hydroxymethyl)-3-mercaptopropanoyl]-L-arginine are obtained.

EXAMPLE 48 N.sup.α -[2-Hydroxymethyl)-3-mercaptopropanoyl]-L-lysine

By substituting 2-(acetoxymethyl)-3-(acetylthio)propanoic acid for the2-(acetylthiomethyl)-3-(acetylthio)propanoic acid in the procedure ofExample 38, and then submitting the product to the procedures ofExamples 39 and 40, N.sup.α-[2-(acetoxymethyl)-3-(acetylthio)propanoyl]-N.sup.ε-tert.butyloxycarbonyl-L-lysine tert.butyl ester, N.sup.α-[2-(acetoxymethyl)-3-(acetylthio)propanoyl]-L-lysine, and N.sup.α-[2-(hydroxymethyl)-3-mercaptopropanoyl]-L-lysine are obtained.

EXAMPLE 49 N-[2-(Hydroxymethyl)-3-mercaptopropanoyl]glycine

By substituting glycine for the L-proline in the procedure of Example19, and then submitting the product to the procedure of Example 20,N-[2-(acetoxymethyl)-3-(acetylthio)propanoyl]glycine andN-[2-(hydroxymethyl)-3-mercaptopropanoyl]glycine are obtained.

EXAMPLE 50 N-[2-(Hydroxymethyl)-3-mercaptopropanoyl]-L-leucine

By substituting L-leucine for the L-proline in the procedure of Example19, and then submitting the product to the procedure of Example 20,N-[2-(acetoxymethyl)-3-(acetylthio)propanoyl]-L-leucine, andN-[2-(hydroxymethyl)-3-mercaptopropanoyl]-L-leucine are obtained.

EXAMPLE 51 N-[2-(Hydroxymethyl-3-mercaptopropanoyl]-L-phenylalanine

By substituting L-phenylalanine for the L-proline in the procedure ofExample 19, and then submitting the product to the procedure of Example20, N-[2-(acetoxymethyl)-3-(acetylthio)propanoyl]-L-phenylalanine andN-[2-(hydroxymethyl)-3-mercaptopropanoyl]-L-phenylalanine are obtained.

EXAMPLE 52 N-[2-(Hydroxymethyl)-3-mercaptopropanoyl]-L-serine

By substituting L-serine for the L-proline in the procedure of Example19, and then submitting the product to the procedure of Example 20,N-[2-(acetoxymethyl)-3-(acetylthio)propanoyl]-L-serine andN-[2-(hydroxymethyl)-3-mercaptopropanoyl]-L-serine are obtained.

EXAMPLE 53 N-[2-(Hydroxymethyl)-3-mercaptopropanoyl]-L-cysteine

By substituting S-ethylcarbamoyl-L-cysteine for the L-proline in theprocedure of Example 19, and then submitting the product to theprocedure of Example 20,N-[2-acetoxymethyl)-3-(acetylthio)propanoyl]-S-ethylcarbamoyl-L-cysteineand N-[2-(hydroxymethyl)-3-mercaptopropanoyl]-L-cysteine are obtained.

EXAMPLE 54 N-[2-(Hydroxymethyl)-3-mercaptopropanoyl]-L-methionine

By substituting L-methionine for the L-proline in the procedure ofExample 19, and then submitting the product to the procedure of Example20, N-[2-(acetoxymethyl)-3-(acetylthio)propanoyl]-L-methionine andN-[2-(hydroxymethyl)-3-mercaptopropanoyl]-L-methionine are obtained.

EXAMPLE 55 N-[2-(Hydroxymethyl)-3-mercaptopropanoyl]-L-asparagine

By substituting L-asparagine for the L-proline in the procedure ofExample 19, and the submitting the product to the procedure of Example20, N-[2-(acetoxymethyl)-3-(acetylthio)propanoyl]-L-asparagine andN-[2-(hydroxymethyl)-3-mercaptopropanoyl]-L-asparagine are obtained.

EXAMPLE 56 N-[2-(Hydroxymethyl)-3-mercaptopropanoyl]-L-glutamic acid

By substituting L-glutamic acid for the L-proline in the procedure ofExample 19, and then submitting the product to the procedure of Example20, N-[2-(acetoxymethyl)-3-(acetylthio)propanoyl]-L-glutamic acid andN-[2-(hydroxymethyl)-3-mercaptopropanoyl]-L-glutamic acid are obtained.

EXAMPLE 57 N-[2-(Hydroxymethyl)-3-mercaptopropanoyl]-L-tyrosine

By substituting L-tyrosine for the L-proline in the procedure of Example19, then submitting the product to the procedure of Example 20,N-[2-(acetoxymethyl)-3-(acetylthiomethyl)propanoyl]-L-tyrosine, andN-[2-(hydroxymethyl)-3-(mercapto)propanoyl]-L-tyrosine are obtained.

EXAMPLE 58N,N'-[Dithiobis(2-hydroxymethyl-3-propanoyl)]-bis-L-phenylalanine

By substitutingN-[2-(hydroxymethyl)-3-mercapto-propanoyl]-L-phenylalanine for the1-[3-mercapto-2-methylthiopropanoyl]-L-proline in the procedure ofExample 30N,N'-[dithiobis(2-hydroxymethyl-3-propanoyl]-bis-L-phenylalanine isobtained.

EXAMPLE 59 2-Acetoxy-3-acetylthiopropanoic acid

By substituting 2-acetoxyacrylic acid for the2-(methylthiomethyl)acrylic acid in the procedure of Example 15,2-acetoxy-3-acetylthiopropanoic acid is obtained.

EXAMPLE 60 N.sup.α -(2-Hydroxy-3-mercaptopropanoyl)-L-lysine

By substituting 2-acetoxy-3-(acetylthio)propanoic acid for the2-(acetylthiomethyl)-3-(acetylthio)propanoic acid in the procedure ofExample 38 and then submitting the product to the procedures of Examples39 and 40, N.sup.α -[2-acetoxy-3-(acetylthio)propanoyl]-N.sup.ε-tert.butyloxycarbonyl-L- lysine tert.butyl ester, N.sup.α-[2-acetoxy-3-(acetylthio)-propanoyl]-L-lysine and N.sup.α-(2-hydroxy-3-mercaptopropanoyl)-L-lysine are obtained.

EXAMPLE 61 1-[(1,2-Dithiolane-4-yl)carbonyl]-L-proline

By substituting 1,2-dithiolane-4-carboxylic acid for the2-(acetylthiomethyl)-3-acetylthiopropanoic acid in the procedure ofExample 2 and then submitting the product to the process of Example 3,1-[(1,2-dithiolane-4-yl)carbonyl]-L-proline tert.butyl ester and1-[(1,2-dithiolane-4-yl)-carbonyl]-L-proline are obtained.

EXAMPLE 62[N-[2-(Hydroxymethyl]-3-mercaptopropanoyl]-N-methyl-L-phenylalanine

By substituting N-methyl-l-phenylalanine for the L-proline in theprocedure of Example 19 and then submitting the product to the procedureof Example 20,N-[2-(acetoxymethyl)-3-(acetylthio)propanoyl]-N-methyl-L-phenylalanineand N-[2-(hydroxymethyl)-3-mercaptopropanoyl]-N-methyl-L-phenylalanineare obtained.

The racemic forms of the final products in each of the foregoingexamples are produced by utilizing the DL-form of the starting aminoacid instead of the L-form.

Similarly the D-form of the final products in each of the foregoingexamples is produced by utilizing the D-form of the starting amino acidinstead of the L-form.

EXAMPLE 63

1000 tablets each containing 100 mg. of1-(2-mercaptomethyl-3-mercaptopropanoyl)-L-proline are produced from thefollowing ingredients:

    ______________________________________                                        1-(2-mercaptomethyl-3-mercapto-                                               propanoyl)-L-Proline     100     g.                                           Corn starch              50      g.                                           Gelatin                  7.5     g.                                           Avicel (microcrystalline cellulose)                                                                    25      g.                                           Magnesium stearate       2.5     g.                                           ______________________________________                                    

The 1-(2-mercaptomethyl-3-mercaptopropanoyl)-L-proline and corn starchare admixed with an aqueous solution of the gelatin. The mixture isdried and ground to a fine powder. The Avicel and then the magnesiumsterate are admixed with the granulation. This is then compressed in atablet to form 1000 tablets each containing 100 g. of active ingredient.

EXAMPLE 64

Two piece #1 gelatin capsules each containing 250 mg. of1-[2,3-dimercaptopropanoyl]-L-proline are filled with a mixture of thefollowing ingredients:

    ______________________________________                                        1-(2,3-dimercaptopropanoyl]-L-proline                                                                  250    mg.                                           Magnesium stearate       7      mg.                                           USP Lactose              193    mg.                                           ______________________________________                                    

EXAMPLE 65

By substituting 1-[2-mercaptomethyl-3-mercaptopropanoyl]-pipecolic acidfor the 1-(2-mercaptomethyl)-3-mercaptopropanoyl)-L-proline in Example63, 1000 tablets each containing 100 mg. of the former are obtained.

What is claimed is:
 1. A compound of the formula ##STR19## and saltsthereof, wherein R is hydrogen or hydroxy;R₁ is hydroxy or lower alkoxy;R₂ is hydrogen, lower alkyl or lower alkanoyl; R₃ is hydrogen or loweralkyl; R₄ is hydrogen, lower alkanoyl, benzoyl or ##STR20## X is oxygenor sulfur; m is 0, 1, 2, 3 or 4; and n is 0 or
 1. 2. A compound as inclaim 1 wherein R is hydrogen; R₁ is hydroxy; R₂ is hydrogen, loweralkyl or lower alkanoyl; R₃ is hydrogen or lower alkyl; R₄ is hydrogen,lower alkanoyl or benzoyl; X is oxygen or sulfur; m is 0 or 1 and n is 0or
 1. 3. A compound as in claim 1 wherein R₁ is hydroxy; R₂, R₃ and R₄each is hydrogen; X is sulfur; and m and n each is
 1. 4. A compound asin claim 1 wherein R₁ is hydroxy; R₂, R₃ and R₄ each is hydrogen; X issulfur; m is 0 and n is
 1. 5. A compound as in claim 1 wherein R ishydrogen.
 6. A compound as in claim 1 wherein R₄ is ##STR21##
 7. Acompound as in claim 1 wherein X is oxygen.
 8. A compound as in claim 1wherein X is sulfur.
 9. A compound of the formula ##STR22## and saltsthereof, wherein R is hydrogen or hydroxy;R₁ is hydroxy or lower alkoxy;R₂ is hydrogen, lower alkyl or lower alkanoyl; R₃ is hydrogen or loweralkyl; R₄ is hydrogen, lower alkanoyl, benzoyl or ##STR23## X is oxygenor sulfur; m is 0, 1, 2, 3 or 4; and n is 0 or
 1. 10. A compound as inclaim 9 wherein R is hydrogen.
 11. A compound as in claim 9 wherein X isoxygen.
 12. A compound as in claim 9 wherein X is sulfur.